Journal: NAR cancer

Article Title: Estrogen receptor alpha mutations regulate gene expression and cell growth in breast cancer through microRNAs.

doi: 10.1093/narcan/zcad027

Figure Lengend Snippet: Figure 5. Inhibition of PRKD3 (PKD3) reduces proliferation in ER WT and mutant cells. ( A ) Bar graphs show three examples of miR-301b altered mutant-specific genes that are likely direct miR-301b target genes, including PRKD3 . Error bars r epr esent average ± SEM for two clones for each genotype. Adjusted P -values are indicated as calculated by DESeq2 (Wald test). ( B ) Diagram of PRKD3 3 ′ UTR indicates the regions with miRNA target sequences. The miR-301b target site is shown in the red box. ( C ) Line graphs show log 2 transformed relati v e confluence ov er time of WT or mutant ER cells treated with anti- PRKD3 siRNAs or negati v e control. Significance between the slopes of the lines was determined using a generalized linear model and the Wald test. ( D ) Relati v e growth is shown for WT, Y537S and D538G ER clones after treatment with CRT0066101 (pan-PKD inhibitor). ( E ) Immunoblot analysis shows PKD3 expression after miR-301b inhibitor treatment in ER WT cells and miR-301b mimic in ER mutant cells. Bar graph shows quantification of PKD3 signal for two clones for each ER genotype. Error bars indicate average signal ± SEM.

Article Snippet: Drug treatments Cells were grown for a single clone for each ER genotype (WT, Y537S and D538G) in 100mm dishes in hormonedepri v ed media for 4 days and then plated in 96-well plates a t approxima tely 12 500 cells per well and allowed to adhere in hormone-depri v ed media for 24 h. Cells were then treated with varying doses (0.01, 0.1, 0.5, 1, 5, 10 or 100 uM) of the PKD inhibitor CRT0066101 (Selleck Chemicals, S8366) or with a vehicle control (H 2 O) and analyzed for proliferation.

Techniques: Inhibition, Mutagenesis, Clone Assay, Transformation Assay, Control, Western Blot, Expressing